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Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists
PROSTAGLANDINS
ARACHIDONATE LIPOXYGENASE INHIBITORS IN GUINEA-PIG ISOLATED TRACHEA. EFFECTS ON CONTRACTIONS TO ANTIGEN AND VARIOUS AGONISTS. A. Yamamoto, K. Shikada and S. Tanaka Shiraoka Research Station of Biological Science, Niaean Chemical Ind. Ltd., Shiraoka, Saitama 349-02, Japan
ABSTRACT We compared the effects of the leukotriene (LT) 04 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase arachidonic acid
(AA),
inhibitor melittin
indomethacin
(5
uM),
and
by
and LTD4. FPL55712 (0.1 and 1 uM) inhibited contractions induced by AA (100 pM) and the phospholipase A2 activator melittin (3 pg/ml). while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 uM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 us/ml) more than by A23187 (1 ug/ml). and these inhibitory effects in the presence of I-serine-borate of FPL55712 were much less complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 pM) had no significant effect on the OA response, whereas the lipoxygenase
inhibitors
I-phenyl-3-pyrazolidone
(phenidone,
10 uM)
and 5.8,11,14-eicosatetraynoic inhibited it. In contrast,
acid (ETYA, 10 uM) clearly NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA. had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig decrease inhibitors
trachea inhibitors of LTD4-induced antigen-induced contractions, whereas reduce the contraction to A23187.
contractions lipoxygenase
INTRODUCTION Leukotrienes (LTs) are implicated to different extents in contractions to antigen and the calcium ionophore A23187 in guinea-pig trachea (I). LTC4. rather than LTD4. is believed to be important in the antisen-induced contractions (2). and LTCn. Dn and E4 play a minor Fole in the A23187-induced &ntraction?-(3): Lipoxygenase inhibitors would therefore be expected to inhibit contractions induced by antigen and A23187 in the guinea-pig trachea more effectively than LTD4 receptor antagonists. The reason why effective concentrations of these lipoxygenase inhibitors is very high, however. may be that they exert effects besides lipoxygenase inhibition. The lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA), phenidone and 5.8,11,14eicosatetraynoic acid (ETYA) inhibited contractions elicited by LTs (4-6). We therefore investigated the effects of low
DECEMBER 1990 VOL. 40 NO. 6
615
concentrations of lipoxygenase inhibitors on antigen- and A23187induced contractions of guinea-pig trachea and compared the results with those of the LTD4 response. AA added in the presence of the cyclooxygenase inhibitor indomethacin cause contractions of guinea-pig trachea (7) that is inhibited by LTD4 receptor antagonists and lipoxygenase inhibitors (8). In the present study, we determined the effective concentrations of the LTD4 receptor antagonist FPL55712 (9) and the 5-lipoxygenase inhibitor NDGA (10) on contraction to the phospholipase A2 (PLA2) activator melittin which release AA (II), in comparison with added AA. Second, in order to investigate the conversion of LTC4 to LTD4, we examined the inhibitory activity of FPL55712 on responses to antigen and A23187 in the presence of I-serine-borate complex (SB), an inhibitor of LTC4 conversion to LTD4 (12); 45 mM SB markedly reduced the ability of FPL55712 to inhibit LTC4-induced contraction of guinea-pig trachea (9.13.14). Finally, we examined the ability of lipoxygenase inhibitors NDGA, phenidone and ETYA to inhibit the antigenand A23187-induced contractions of the guinea-pig trachea, in comparison with the response to LTD4. METHODS Male Hartley guinea-pigs (200-250 g) were sensitized with ovalbumin (OA) 100 mg subcutaneously and 100 mg intraperitoneally. The trachea was removed 3-4 weeks later, cut spirally, and divided into three or four equal segments (one serving as the control). In a separate experiment tracheas were removed from non-sensitized guinea-pigs (300-400 g). Tissues were suspended under 1 g tension in 8 ml organ baths containing modified Tyrode's solution, maintained at 37°C. and bubbled with 02/CO2(95:5). The composition of the modified Tyrode's solution was (r&l): NaCl 137, KC1 2.7, CaC12 1.8, MgC12 1.0, NaH2P04 0.3, NaHC03 20 and dextrose Il. Contractions of the tracheal strips were measured using an isotonic transducer (Type TD-112S, Nihon Kohden). Tissues were equilibrated for 50-60 min and then the maximal response to 100 uM histamine was recorded. Tissues were washed several times over a 30 min period until the resting tone level was restored, and then incubated for 30 min with various inhibitors in the presence of 5 uM indomethacin. DATA ANALYSIS The contractile responses of the tracheal strips were measured as percentages of the maximum response obtained with 100 uM histamine for each specimen, and compared using two methods: 1) the area under the contraction curve, which was calculated using a computer program, and 2) the contraction height 30 min after the challenge. The data are presented as means 2 s.e. mean. Data for the area under the contraction curve and contraction height were analyzed for significance using a two-tailed Student's t-test for paired data. Values of P < 0.05 were regarded as significant.
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DECEMBER199OVOL.4ONO.6
PROSTAGLANDINS DRUGS OA (grade II for sensitization AA (sodium salt), melittin. and grade III for challenge), calcium ionophore A23187, NDGA, phenidone and indomethacin were purchased from Sigma (St. Louis. ETYA, LTD4-monomethylester and histamine were purchased from MO). Fluka (Switzerland), Paesel (West Germany) and Wako (Japan), respectively. FPL55712 was synthesized in our laboratory. RESULTS
In the presence of 5 uM indomethacin. 100 PM AA caused a Its slow-onset, sustained contraction of the guinea-pig trachea. magnitude 30 min after adding the AA was about 35-45% of the histamine maximum. The LTD4 receptor antagonist FPL55712 0.1 and 1 uM. and the lipoxygenase inhibitor NDGA 10 uM inhibited the AA response (Fig. 1 a.c, Table 1). FPL55712 was more potent and effective than NDGA in inhibiting the AA response, judged both by the areas under the contraction curves and the height at 30 min.
AA 50
s = F!
0
/i
I, NDGA
Time (min)
Fig. 1 Effects of FPL55712 (a,b) or NDGA (c,d) on contractions induced by AA (100 uM)(a.c) or melittin (3 ug/ml)(b,d) in guinea-pig trachea. Responses are expressed as percentages of contractions induced by 100 uM histamine. Each point represents the mean + s.e. mean of 4-5 experiments. Symbols indicate the absence ( l ) and the presence of inhibitor 0.1 uM ( 0 ), 1 uM ( A ) or 10 uM ( 0). * P -C 0.05, compared to time-matched controls.
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PROSTAGLANDINS Table 1 Effects of FPL55712 or NDGA on contractions of trachea induced by AA (100 uM) or melittin (3 ug/ml)
Area FPL55712 0.1 uM 1 uM
83 + 7 41 z 104
AA Height
at 30 min
77 + 8" 29 z 8-y
guinea-pig
Melittin Height at 30 min
Area
76 + 15 46 2 14*
74 + 14 47 I 10"
75 t 19 1 i 1"
82 t 19 0 2 0"
NDGA
1 uM 10 uM
134 t 19 81 g 7*
110 t 11 72 z 9"
Values are mean % (+ s.e. mean) of the control * P < 0.05, compare;j to control.
values.
The PLA2 activator melittin (3 ug/ml) also produced a slowly developing contraction which at 30 min was about 40% of the histamine maximum. FPL55712 0.1 and 1 pM or NDGA 1 and 10 uM concentration-dependently inhibited the melittin response (Fig. 1 b,d), as shown both by the areas under the contraction curves and the height at 30 min. NDGA was more effective against melittin than against AA (Table 1). Sensitized trachea challenged with OA (100 us/ml) responded with a sustained contraction that usually recorded its peak within 5 to 10 min. The early phase of the OA response was not affected by FPL55712 (1 uM), but the latter phase was clearly inhibited (Fig. Since FPL55712 did not affect the early phase of the OA 2 a). response, the inhibitory effect of FPL55712 on the area under the contraction curve was less pronounced than on the height at 30 min (Table 2). Contractile responses induced by OA were not altered by 45 mM SB, which inhibits the conversion of LTC4 to LTD4, but the inhibitory effects of FPL55712 on the OA response tended to be less (P=O.lO for the area, P=O.O4 for the height at 30 min) (Fig. 2 b, Table 2). The calcium ionophore A23187 (1 us/ml) produced a slow-onset contraction in non-sensitized tracheas, and maximal responses were not obtained within 30 min. FPL55712 1 uM tended to reduce the A23187 response (P = 0.11 for the area, P=O.12 for the height at 30 min) (Fig. 2 c. Table 2). The efficacy of FPL55712 in inhibiting the contraction height at 30 min was greater for OA than for A23187. SB 45 mM did not alter the control A23187 but it tended to reduce the inhibitory effect of responses, FPL55712 (P=O.13 for the area, P=O.O7 for the height at 30 min).
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DECEMBER 1990 VOL. 40 NO. 6
PROSTAGLANDINS +SB
Time (mln)
Fig.
2
Effects of FPL55712 on contractions induced by OA 100 ug/mI (a,b) and A23187 I ug/mI (c,d) in the absence (a.c) and the presence (b,d) of SB 45 mM in guinea-pig trachea. Responses are expressed as percentages of contractions induced by 100 uM histamine. Each Symbols point represents the mean f. s.e. mean of 5 experiments. indicate
the
(0) or controls.
I
Table 2 Effects of by OA 100 of
SB
45
absence uM
( l ) and the presence of * P < 0.05, compared
(n).
FPL55712 on contractions of ug/ml and A23187 I ug/ml
FPL55712 0.1 uM to time-matched
guinea-pig trachea in the absence and
induced presence
mM no SB Height at
Area
45 30 min
Area
mM SB Height
at
30 min
OA FPL55712 0.1 uM 1 I.IM
90 + 6 57 z 7"
78 + 9 35 z IO"
NT 88+
I6
NT 84 +
18t
II4 +
I4
109 +
IO
A23187 FPL55712 1 uM
88 +
7
82 +
Values are mean % (2 s.e. mean * P < 0.05, NT: Not tested, compared to no SB.
DECEMBER 1990 VOL. 40 NO. 6
9
) of the compared
control values. to control, t
P
<
0.05,
619
PROSTAGLANDINS NDGA 10 uM had no significant effect on the OA response, but tended to reduce that to A23187 (Fig. 3 a.d: Table 3). Phenidone moderately reduced both OA and A23187 responses (Fig. 3 b,e; Table 3). ETYA markedly inhibited the OA response, but it had no significant effect on that to A23187 (Fig. 3 c,f). Both phenidone and ETYA inhibited only the latter phase of the OA response, as occurred with FPL55712. The order of effectiveness in inhibiting the OA response was FPL55712 > ETYA > phenidone >> NOGA.
ETYA
Time
(mln)
Fig.3 Effects of NDGA (a,d), phenidone (b,e) and ETYA (c.f) 10 uM on contractions induced by OA 100 ug/ml (a,b.c) and A23187 1 ug/ml in guinea-pig trachea. Responses are expressed as (d .e,f) percentages of contractions to 100 uM histamine. Each point represents the mean + s.e. mean of 4-6 experiments. Symbols indicate the absence ( l > and presence of inhibitor ( o ). * P < 0.05, compared to time-matched controls.
Phenidone and ETYA 10 pM markedly inhibited LTD4-induced contraction of the guinea-pig trachea, but NDGA 10 uM had no effect on it (Fig. 4). The order of potency in inhibiting the LTD4 response was FPL55712 z-> ETYA > phenidone >> NDGA = 0. similar to the results with OA. These inhibitors did not reduce histamine-induced submaximal contraction (10 uM histamine) of the guinea-pig trachea (Fig. 5).
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DECEMBER 1990 VOL. 40 NO. 6
PROSTAGLANDINS Table 3 Effects of NDGA, by OA 100 ug/ml
Stimulus
_~
phenidone and ETYA 70 uM on contractions and A23187 1 ug/ml
ETYA
Phenidone
NDGA
~~
induced
OA
a7 + 15
79 + 4"
48 + 13"
A23187
77 + 11
71 2 5"
86 + 12
Values are mean % (+ s.e. mean) of the control values for the area under the contraction curve, * P < 0.05. compared to control.
NDGA
FPL55712 b
100 a A
I
I
A3
L
E ;
Phenidone
ETYA
loo-,
.d
ap +/+ 50.
/ f
,*/+‘+ ,o*
-Log LTD4
(M)
E$,c& of FPL55712 (a), NDGA (b), phenidone (c) and ETYA (d) on Responses are LTD4-induced contractions of guinea-pig trachea. expressed as percentages of contractions induced by 100 uM Each point represents the mean + s.e. mean of 3-4 histamine. Symbols indicate the absence ( l ) and presence of experiments. inhibitor 0.1 uM ( 0 ). 1 uM ( A) or 10 uM ( o >. * P < 0.05, compared to corresponding controls.
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PROSTAGLANDINS
NDGA
FPL55712
ETYA
Phenldone 100
d .
! -1p-a-=Q i
r
-Log Histamine
Fig.
J:. 6
5
(M)
5
Effects of FPL55712 (a), NDGA (b). phenidone (c), and ETYA (d) on histamine-induced contractions of guinea-pig trachea. Responses are expressed as percentages of contractions induced by 100 uM histamine. Each point represents the mean + s.e. mean of 4-5 experiments. Symbols indicate the absence ( l ) and presence of inhibitor 1 uM ( A) or IO uM ( o ). * P < 0.05. compared to corresponding controls.
DISCUSSION
In the presence of the cyclooxygenase inhibitor indomethacin, exogenous AA elicits contractions of the guinea-pig trachea (8) In the present study, the LTD4 which are largely mediated by LTs. AA- and melittin-induced receptor antagonist FPL55712 inhibited contractions to similar extents. These results suggest that LTs are involved in contractions induced by melittin. The melittin lipoxygenase inhibitor NDGA more effectively inhibited than AA, indicating a greater effectiveness against endogenously released AA. The melittin-induced contraction is a good model for evaluation of inhibitors affecting the metabolism of endogenously generated AA in the lipoxygenase pathway. The early and later phases of the OA response are mediated respectively by histamine and peptido-LTs (15). In the present selective LTD4 receptor antagonist FPL55712 did not study, the affect the early phase of the OA response but greatly inhibited The inhibition of both OA and A23187 responses the latter phase. by FPL55712 were much less in the presence of SB, which inhibits the conversion of LTC4 to LTD4 in the guinea-pig trachea (12). We
622
DECEMBER 199OVOL. 40 NO. 6
PROSTAGLANDINS LTD4, and possibly suggest that the LTC4 metabolite therefore LTE4, are involved in both OA- and A23187-induced contractions of and that the contribution of LTD4 to the the guinea-pig trachea, A23187 response is less than to the OA response. inhibited the melittin-induced NDGA 10 uM almost completely but had no significant effect on the OA response. contractions, The inability of NDGA to inhibit the OA response is not due to the magnitude of the contraction induced by OA, since FPL55712 had the same inhibitory effect on both the OA response (the latter phase) other lipoxygenase In contrast, and the melittin response. inhibited the OA inhibitors, i.e. phenidone and ETYA, clearly phenidone and ETYA, but not NDGA, response. However, both Thus the decrease greatly reduced the LTD4-induced contraction. of the OA response by phenidone and ETYA presumably depend on preventing the LTD4 response by phenidone and ETYA rather than a block of lipoxygenase; NDGA did not affect the OA response prevent the LTD4 response in the presumably because it does not guinea-pig trachea. The reduction of the A23187 response by NDGA and phenidone may NDGA reduced be due to a block of the AA lipoxygenase, because the A23187 response without affecting LTD4, while phenidone produced about equally marked reduction of the LTD4 response and of A23187 and OA. In contrast, the LTD4 receptor antagonist FPL55712 was less effective on the A23187 response than on those of OA and LTD4. From these results, it appears that inhibitors of contractions induced by LTD4 decrease the OA response, while lipoxygenase inhibitors reduce the A23187 response in the guineapig trachea. ETYA inhibits the AA lipoxygenase pathway in human leukocytes and platelets (IO), and is more potent than NDGA in inhibiting SRS-A generation from sensitized guinea-pig lung The reason for the inability of ETYA to inhibit fragments (16). species the A23187 response is not clear but may be related to (17) or tissue differences. In conclusion, the present data suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.
REFERENCES 1.
Burka. J.F: & Paterson, N.A.M. A comparison of antigeninduced and calcium ionophore A23187-induced contraction of isolated guinea pig trachea. Can. J. Physiol. Pharmacol. 2: 1031, 1981.
2.
Jones, T.R., Charette, L. & Denis, D. Antigen-induced contraction of guinea-pig trachea: studies with novel inhibitors and antagonists of arachidonic acid metabolites. Br. J. Pharmac. 95: 309, 1988.
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PROSTAGLANDINS
3.
Bur‘ka, J.F. Pharmacological modulation of responses of guinea-pig airways contracted with antigen and calcium ionophore A23187. Br. J. Pharmac. 85: 411, 1985.
4.
Hageman. W.E., Rose, M.P. & Persico, F.J. Antagonism by ETYA of the effects of leukotrienes on ileum and lung parenchymal strips independent of effects on arachidonic acid metabolism. Prostaglandins -32: 563, 1986.
5.
Schwalm, S.F., Lewis, A.J. & Hand, J.M. Inhibition of leukotriene-induced contraction of guinea pig trachea by 5lipoxygenase inhibitors. Prostaglandins 3: 113, 1987.
6.
Shikada, K., Yamamoto, A. & Tanaka, S. Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea. Prostaglandins 533, 1988. -36:
7.
Burka, J.F. Effects of indomethacin on airway contraction and the release of LTC4-like material. Prostaglandins 29: 529, 1985.
8.
Burka, J.F. Pharmacological guinea-pig airways contracted Pharmac. -85: 421, 1985.
9.
Pharmacological evidence for a Snyder, O.W. & Krell. R.O. distinct leukotriene C4 receptor in guinea-pig trachea. J. Pharmacol. Exp. Ther. -231: 616, 1984.
10.
Salari, H., Braquet. P. & Borgeat, P. Comparative effects of indomethacin, acetylenic acids, 15-HETE, nordihydroguaiaretic acid and BW755C on the metabolism of arachidonic acid in human leukocytes and platelets. Prostaglandins Leukotrienes Med. 2: 53, 1984.
11.
Shier, W.T. Activation of high phospholipase A2 in cultured cells. U.S.A. -76: 195, 1979.
12.
Snyder, D.W., Aharony, D., Dobson, P., Tsai, B.S. & Krell, R.D. Pharmacological and biological evidence for metabolism of peptide leukotrienes by guinea-pig airway smooth muscle in vitro. J. Pharmacol. Exp. Ther. -231: 224, 1984.
13.
Charette, L. & Jones, T.R. Effects of L-serine antagonism of leukotriene C4-induced contractions pig trachea. Br. J. Pharmac. 91: 179. 1987.
14.
Muccitelli, R.M., Tucker, S.S., Hay, D.W.P.. Torphy, T.J. & Wasserman, M.A. Is the guinea pig trachea a good in vitro model of human large and central airways ?. Comparison on leukotriene-, methacholine-, histamineand antigen-induced contractions. J. Pharmacol. Exp. Ther. -243: 467, 1987.
624
modulation of with arachidonic
levels Proc.
responses of acid. Br. J.
of endogenous Natl. Acad. Sci.
borate on of guinea-
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15.
Adams, G.K. & Lichtenstein, L. In vitro studies of antigeninduced bronchospasm: Effect of antihistamine and SRS-A antagonist on response of sensitized guinea pig and human airways to antigen. J. Immunol. -122: 555, 1979.
16.
Ashida, Y., Saijo, T.. Kuriki, H., Makino. Maki, Y. Pharmacological profile of AA-861, inhibitor. Prostaglandins -26: 955. 1983.
17.
Okumura, M., Obata, N., Yamamura, H.. Kohno. S. & Ohata, K. Species difference of 5-lipoxygenase derived from polymorphonuclear leukocytes on sensitivity to drugs. Japn. J. Pharmacol. 37, 1989. -50:
Editor:
A.
Bennett
DECEMBER 1990VOL. 40 NO. 6
Received
2-22-90
H., Terao. S. & a 5-lipoxygenase
Accepted
g-26-90
ARACHIDONATE LIPOXYGENASE INHIBITORS IN GUINEA-PIG ISOLATED TRACHEA. EFFECTS ON CONTRACTIONS TO ANTIGEN AND VARIOUS AGONISTS. A. Yamamoto, K. Shikada and S. Tanaka Shiraoka Research Station of Biological Science, Niaean Chemical Ind. Ltd., Shiraoka, Saitama 349-02, Japan
ABSTRACT We compared the effects of the leukotriene (LT) 04 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase arachidonic acid
(AA),
inhibitor melittin
indomethacin
(5
uM),
and
by
and LTD4. FPL55712 (0.1 and 1 uM) inhibited contractions induced by AA (100 pM) and the phospholipase A2 activator melittin (3 pg/ml). while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 uM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 us/ml) more than by A23187 (1 ug/ml). and these inhibitory effects in the presence of I-serine-borate of FPL55712 were much less complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 pM) had no significant effect on the OA response, whereas the lipoxygenase
inhibitors
I-phenyl-3-pyrazolidone
(phenidone,
10 uM)
and 5.8,11,14-eicosatetraynoic inhibited it. In contrast,
acid (ETYA, 10 uM) clearly NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA. had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig decrease inhibitors
trachea inhibitors of LTD4-induced antigen-induced contractions, whereas reduce the contraction to A23187.
contractions lipoxygenase
INTRODUCTION Leukotrienes (LTs) are implicated to different extents in contractions to antigen and the calcium ionophore A23187 in guinea-pig trachea (I). LTC4. rather than LTD4. is believed to be important in the antisen-induced contractions (2). and LTCn. Dn and E4 play a minor Fole in the A23187-induced &ntraction?-(3): Lipoxygenase inhibitors would therefore be expected to inhibit contractions induced by antigen and A23187 in the guinea-pig trachea more effectively than LTD4 receptor antagonists. The reason why effective concentrations of these lipoxygenase inhibitors is very high, however. may be that they exert effects besides lipoxygenase inhibition. The lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA), phenidone and 5.8,11,14eicosatetraynoic acid (ETYA) inhibited contractions elicited by LTs (4-6). We therefore investigated the effects of low
DECEMBER 1990 VOL. 40 NO. 6
615
concentrations of lipoxygenase inhibitors on antigen- and A23187induced contractions of guinea-pig trachea and compared the results with those of the LTD4 response. AA added in the presence of the cyclooxygenase inhibitor indomethacin cause contractions of guinea-pig trachea (7) that is inhibited by LTD4 receptor antagonists and lipoxygenase inhibitors (8). In the present study, we determined the effective concentrations of the LTD4 receptor antagonist FPL55712 (9) and the 5-lipoxygenase inhibitor NDGA (10) on contraction to the phospholipase A2 (PLA2) activator melittin which release AA (II), in comparison with added AA. Second, in order to investigate the conversion of LTC4 to LTD4, we examined the inhibitory activity of FPL55712 on responses to antigen and A23187 in the presence of I-serine-borate complex (SB), an inhibitor of LTC4 conversion to LTD4 (12); 45 mM SB markedly reduced the ability of FPL55712 to inhibit LTC4-induced contraction of guinea-pig trachea (9.13.14). Finally, we examined the ability of lipoxygenase inhibitors NDGA, phenidone and ETYA to inhibit the antigenand A23187-induced contractions of the guinea-pig trachea, in comparison with the response to LTD4. METHODS Male Hartley guinea-pigs (200-250 g) were sensitized with ovalbumin (OA) 100 mg subcutaneously and 100 mg intraperitoneally. The trachea was removed 3-4 weeks later, cut spirally, and divided into three or four equal segments (one serving as the control). In a separate experiment tracheas were removed from non-sensitized guinea-pigs (300-400 g). Tissues were suspended under 1 g tension in 8 ml organ baths containing modified Tyrode's solution, maintained at 37°C. and bubbled with 02/CO2(95:5). The composition of the modified Tyrode's solution was (r&l): NaCl 137, KC1 2.7, CaC12 1.8, MgC12 1.0, NaH2P04 0.3, NaHC03 20 and dextrose Il. Contractions of the tracheal strips were measured using an isotonic transducer (Type TD-112S, Nihon Kohden). Tissues were equilibrated for 50-60 min and then the maximal response to 100 uM histamine was recorded. Tissues were washed several times over a 30 min period until the resting tone level was restored, and then incubated for 30 min with various inhibitors in the presence of 5 uM indomethacin. DATA ANALYSIS The contractile responses of the tracheal strips were measured as percentages of the maximum response obtained with 100 uM histamine for each specimen, and compared using two methods: 1) the area under the contraction curve, which was calculated using a computer program, and 2) the contraction height 30 min after the challenge. The data are presented as means 2 s.e. mean. Data for the area under the contraction curve and contraction height were analyzed for significance using a two-tailed Student's t-test for paired data. Values of P < 0.05 were regarded as significant.
616
DECEMBER199OVOL.4ONO.6
PROSTAGLANDINS DRUGS OA (grade II for sensitization AA (sodium salt), melittin. and grade III for challenge), calcium ionophore A23187, NDGA, phenidone and indomethacin were purchased from Sigma (St. Louis. ETYA, LTD4-monomethylester and histamine were purchased from MO). Fluka (Switzerland), Paesel (West Germany) and Wako (Japan), respectively. FPL55712 was synthesized in our laboratory. RESULTS
In the presence of 5 uM indomethacin. 100 PM AA caused a Its slow-onset, sustained contraction of the guinea-pig trachea. magnitude 30 min after adding the AA was about 35-45% of the histamine maximum. The LTD4 receptor antagonist FPL55712 0.1 and 1 uM. and the lipoxygenase inhibitor NDGA 10 uM inhibited the AA response (Fig. 1 a.c, Table 1). FPL55712 was more potent and effective than NDGA in inhibiting the AA response, judged both by the areas under the contraction curves and the height at 30 min.
AA 50
s = F!
0
/i
I, NDGA
Time (min)
Fig. 1 Effects of FPL55712 (a,b) or NDGA (c,d) on contractions induced by AA (100 uM)(a.c) or melittin (3 ug/ml)(b,d) in guinea-pig trachea. Responses are expressed as percentages of contractions induced by 100 uM histamine. Each point represents the mean + s.e. mean of 4-5 experiments. Symbols indicate the absence ( l ) and the presence of inhibitor 0.1 uM ( 0 ), 1 uM ( A ) or 10 uM ( 0). * P -C 0.05, compared to time-matched controls.
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617
PROSTAGLANDINS Table 1 Effects of FPL55712 or NDGA on contractions of trachea induced by AA (100 uM) or melittin (3 ug/ml)
Area FPL55712 0.1 uM 1 uM
83 + 7 41 z 104
AA Height
at 30 min
77 + 8" 29 z 8-y
guinea-pig
Melittin Height at 30 min
Area
76 + 15 46 2 14*
74 + 14 47 I 10"
75 t 19 1 i 1"
82 t 19 0 2 0"
NDGA
1 uM 10 uM
134 t 19 81 g 7*
110 t 11 72 z 9"
Values are mean % (+ s.e. mean) of the control * P < 0.05, compare;j to control.
values.
The PLA2 activator melittin (3 ug/ml) also produced a slowly developing contraction which at 30 min was about 40% of the histamine maximum. FPL55712 0.1 and 1 pM or NDGA 1 and 10 uM concentration-dependently inhibited the melittin response (Fig. 1 b,d), as shown both by the areas under the contraction curves and the height at 30 min. NDGA was more effective against melittin than against AA (Table 1). Sensitized trachea challenged with OA (100 us/ml) responded with a sustained contraction that usually recorded its peak within 5 to 10 min. The early phase of the OA response was not affected by FPL55712 (1 uM), but the latter phase was clearly inhibited (Fig. Since FPL55712 did not affect the early phase of the OA 2 a). response, the inhibitory effect of FPL55712 on the area under the contraction curve was less pronounced than on the height at 30 min (Table 2). Contractile responses induced by OA were not altered by 45 mM SB, which inhibits the conversion of LTC4 to LTD4, but the inhibitory effects of FPL55712 on the OA response tended to be less (P=O.lO for the area, P=O.O4 for the height at 30 min) (Fig. 2 b, Table 2). The calcium ionophore A23187 (1 us/ml) produced a slow-onset contraction in non-sensitized tracheas, and maximal responses were not obtained within 30 min. FPL55712 1 uM tended to reduce the A23187 response (P = 0.11 for the area, P=O.12 for the height at 30 min) (Fig. 2 c. Table 2). The efficacy of FPL55712 in inhibiting the contraction height at 30 min was greater for OA than for A23187. SB 45 mM did not alter the control A23187 but it tended to reduce the inhibitory effect of responses, FPL55712 (P=O.13 for the area, P=O.O7 for the height at 30 min).
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DECEMBER 1990 VOL. 40 NO. 6
PROSTAGLANDINS +SB
Time (mln)
Fig.
2
Effects of FPL55712 on contractions induced by OA 100 ug/mI (a,b) and A23187 I ug/mI (c,d) in the absence (a.c) and the presence (b,d) of SB 45 mM in guinea-pig trachea. Responses are expressed as percentages of contractions induced by 100 uM histamine. Each Symbols point represents the mean f. s.e. mean of 5 experiments. indicate
the
(0) or controls.
I
Table 2 Effects of by OA 100 of
SB
45
absence uM
( l ) and the presence of * P < 0.05, compared
(n).
FPL55712 on contractions of ug/ml and A23187 I ug/ml
FPL55712 0.1 uM to time-matched
guinea-pig trachea in the absence and
induced presence
mM no SB Height at
Area
45 30 min
Area
mM SB Height
at
30 min
OA FPL55712 0.1 uM 1 I.IM
90 + 6 57 z 7"
78 + 9 35 z IO"
NT 88+
I6
NT 84 +
18t
II4 +
I4
109 +
IO
A23187 FPL55712 1 uM
88 +
7
82 +
Values are mean % (2 s.e. mean * P < 0.05, NT: Not tested, compared to no SB.
DECEMBER 1990 VOL. 40 NO. 6
9
) of the compared
control values. to control, t
P
<
0.05,
619
PROSTAGLANDINS NDGA 10 uM had no significant effect on the OA response, but tended to reduce that to A23187 (Fig. 3 a.d: Table 3). Phenidone moderately reduced both OA and A23187 responses (Fig. 3 b,e; Table 3). ETYA markedly inhibited the OA response, but it had no significant effect on that to A23187 (Fig. 3 c,f). Both phenidone and ETYA inhibited only the latter phase of the OA response, as occurred with FPL55712. The order of effectiveness in inhibiting the OA response was FPL55712 > ETYA > phenidone >> NOGA.
ETYA
Time
(mln)
Fig.3 Effects of NDGA (a,d), phenidone (b,e) and ETYA (c.f) 10 uM on contractions induced by OA 100 ug/ml (a,b.c) and A23187 1 ug/ml in guinea-pig trachea. Responses are expressed as (d .e,f) percentages of contractions to 100 uM histamine. Each point represents the mean + s.e. mean of 4-6 experiments. Symbols indicate the absence ( l > and presence of inhibitor ( o ). * P < 0.05, compared to time-matched controls.
Phenidone and ETYA 10 pM markedly inhibited LTD4-induced contraction of the guinea-pig trachea, but NDGA 10 uM had no effect on it (Fig. 4). The order of potency in inhibiting the LTD4 response was FPL55712 z-> ETYA > phenidone >> NDGA = 0. similar to the results with OA. These inhibitors did not reduce histamine-induced submaximal contraction (10 uM histamine) of the guinea-pig trachea (Fig. 5).
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DECEMBER 1990 VOL. 40 NO. 6
PROSTAGLANDINS Table 3 Effects of NDGA, by OA 100 ug/ml
Stimulus
_~
phenidone and ETYA 70 uM on contractions and A23187 1 ug/ml
ETYA
Phenidone
NDGA
~~
induced
OA
a7 + 15
79 + 4"
48 + 13"
A23187
77 + 11
71 2 5"
86 + 12
Values are mean % (+ s.e. mean) of the control values for the area under the contraction curve, * P < 0.05. compared to control.
NDGA
FPL55712 b
100 a A
I
I
A3
L
E ;
Phenidone
ETYA
loo-,
.d
ap +/+ 50.
/ f
,*/+‘+ ,o*
-Log LTD4
(M)
E$,c& of FPL55712 (a), NDGA (b), phenidone (c) and ETYA (d) on Responses are LTD4-induced contractions of guinea-pig trachea. expressed as percentages of contractions induced by 100 uM Each point represents the mean + s.e. mean of 3-4 histamine. Symbols indicate the absence ( l ) and presence of experiments. inhibitor 0.1 uM ( 0 ). 1 uM ( A) or 10 uM ( o >. * P < 0.05, compared to corresponding controls.
DECEMBER
19% VOL. 40 NO. 6
621
PROSTAGLANDINS
NDGA
FPL55712
ETYA
Phenldone 100
d .
! -1p-a-=Q i
r
-Log Histamine
Fig.
J:. 6
5
(M)
5
Effects of FPL55712 (a), NDGA (b). phenidone (c), and ETYA (d) on histamine-induced contractions of guinea-pig trachea. Responses are expressed as percentages of contractions induced by 100 uM histamine. Each point represents the mean + s.e. mean of 4-5 experiments. Symbols indicate the absence ( l ) and presence of inhibitor 1 uM ( A) or IO uM ( o ). * P < 0.05. compared to corresponding controls.
DISCUSSION
In the presence of the cyclooxygenase inhibitor indomethacin, exogenous AA elicits contractions of the guinea-pig trachea (8) In the present study, the LTD4 which are largely mediated by LTs. AA- and melittin-induced receptor antagonist FPL55712 inhibited contractions to similar extents. These results suggest that LTs are involved in contractions induced by melittin. The melittin lipoxygenase inhibitor NDGA more effectively inhibited than AA, indicating a greater effectiveness against endogenously released AA. The melittin-induced contraction is a good model for evaluation of inhibitors affecting the metabolism of endogenously generated AA in the lipoxygenase pathway. The early and later phases of the OA response are mediated respectively by histamine and peptido-LTs (15). In the present selective LTD4 receptor antagonist FPL55712 did not study, the affect the early phase of the OA response but greatly inhibited The inhibition of both OA and A23187 responses the latter phase. by FPL55712 were much less in the presence of SB, which inhibits the conversion of LTC4 to LTD4 in the guinea-pig trachea (12). We
622
DECEMBER 199OVOL. 40 NO. 6
PROSTAGLANDINS LTD4, and possibly suggest that the LTC4 metabolite therefore LTE4, are involved in both OA- and A23187-induced contractions of and that the contribution of LTD4 to the the guinea-pig trachea, A23187 response is less than to the OA response. inhibited the melittin-induced NDGA 10 uM almost completely but had no significant effect on the OA response. contractions, The inability of NDGA to inhibit the OA response is not due to the magnitude of the contraction induced by OA, since FPL55712 had the same inhibitory effect on both the OA response (the latter phase) other lipoxygenase In contrast, and the melittin response. inhibited the OA inhibitors, i.e. phenidone and ETYA, clearly phenidone and ETYA, but not NDGA, response. However, both Thus the decrease greatly reduced the LTD4-induced contraction. of the OA response by phenidone and ETYA presumably depend on preventing the LTD4 response by phenidone and ETYA rather than a block of lipoxygenase; NDGA did not affect the OA response prevent the LTD4 response in the presumably because it does not guinea-pig trachea. The reduction of the A23187 response by NDGA and phenidone may NDGA reduced be due to a block of the AA lipoxygenase, because the A23187 response without affecting LTD4, while phenidone produced about equally marked reduction of the LTD4 response and of A23187 and OA. In contrast, the LTD4 receptor antagonist FPL55712 was less effective on the A23187 response than on those of OA and LTD4. From these results, it appears that inhibitors of contractions induced by LTD4 decrease the OA response, while lipoxygenase inhibitors reduce the A23187 response in the guineapig trachea. ETYA inhibits the AA lipoxygenase pathway in human leukocytes and platelets (IO), and is more potent than NDGA in inhibiting SRS-A generation from sensitized guinea-pig lung The reason for the inability of ETYA to inhibit fragments (16). species the A23187 response is not clear but may be related to (17) or tissue differences. In conclusion, the present data suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.
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Burka. J.F: & Paterson, N.A.M. A comparison of antigeninduced and calcium ionophore A23187-induced contraction of isolated guinea pig trachea. Can. J. Physiol. Pharmacol. 2: 1031, 1981.
2.
Jones, T.R., Charette, L. & Denis, D. Antigen-induced contraction of guinea-pig trachea: studies with novel inhibitors and antagonists of arachidonic acid metabolites. Br. J. Pharmac. 95: 309, 1988.
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PROSTAGLANDINS
3.
Bur‘ka, J.F. Pharmacological modulation of responses of guinea-pig airways contracted with antigen and calcium ionophore A23187. Br. J. Pharmac. 85: 411, 1985.
4.
Hageman. W.E., Rose, M.P. & Persico, F.J. Antagonism by ETYA of the effects of leukotrienes on ileum and lung parenchymal strips independent of effects on arachidonic acid metabolism. Prostaglandins -32: 563, 1986.
5.
Schwalm, S.F., Lewis, A.J. & Hand, J.M. Inhibition of leukotriene-induced contraction of guinea pig trachea by 5lipoxygenase inhibitors. Prostaglandins 3: 113, 1987.
6.
Shikada, K., Yamamoto, A. & Tanaka, S. Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea. Prostaglandins 533, 1988. -36:
7.
Burka, J.F. Effects of indomethacin on airway contraction and the release of LTC4-like material. Prostaglandins 29: 529, 1985.
8.
Burka, J.F. Pharmacological guinea-pig airways contracted Pharmac. -85: 421, 1985.
9.
Pharmacological evidence for a Snyder, O.W. & Krell. R.O. distinct leukotriene C4 receptor in guinea-pig trachea. J. Pharmacol. Exp. Ther. -231: 616, 1984.
10.
Salari, H., Braquet. P. & Borgeat, P. Comparative effects of indomethacin, acetylenic acids, 15-HETE, nordihydroguaiaretic acid and BW755C on the metabolism of arachidonic acid in human leukocytes and platelets. Prostaglandins Leukotrienes Med. 2: 53, 1984.
11.
Shier, W.T. Activation of high phospholipase A2 in cultured cells. U.S.A. -76: 195, 1979.
12.
Snyder, D.W., Aharony, D., Dobson, P., Tsai, B.S. & Krell, R.D. Pharmacological and biological evidence for metabolism of peptide leukotrienes by guinea-pig airway smooth muscle in vitro. J. Pharmacol. Exp. Ther. -231: 224, 1984.
13.
Charette, L. & Jones, T.R. Effects of L-serine antagonism of leukotriene C4-induced contractions pig trachea. Br. J. Pharmac. 91: 179. 1987.
14.
Muccitelli, R.M., Tucker, S.S., Hay, D.W.P.. Torphy, T.J. & Wasserman, M.A. Is the guinea pig trachea a good in vitro model of human large and central airways ?. Comparison on leukotriene-, methacholine-, histamineand antigen-induced contractions. J. Pharmacol. Exp. Ther. -243: 467, 1987.
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modulation of with arachidonic
levels Proc.
responses of acid. Br. J.
of endogenous Natl. Acad. Sci.
borate on of guinea-
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15.
Adams, G.K. & Lichtenstein, L. In vitro studies of antigeninduced bronchospasm: Effect of antihistamine and SRS-A antagonist on response of sensitized guinea pig and human airways to antigen. J. Immunol. -122: 555, 1979.
16.
Ashida, Y., Saijo, T.. Kuriki, H., Makino. Maki, Y. Pharmacological profile of AA-861, inhibitor. Prostaglandins -26: 955. 1983.
17.
Okumura, M., Obata, N., Yamamura, H.. Kohno. S. & Ohata, K. Species difference of 5-lipoxygenase derived from polymorphonuclear leukocytes on sensitivity to drugs. Japn. J. Pharmacol. 37, 1989. -50:
Editor:
A.
Bennett
DECEMBER 1990VOL. 40 NO. 6
Received
2-22-90
H., Terao. S. & a 5-lipoxygenase
Accepted
g-26-90